S269: MACROPHAGE FUNCTIONAL AND METABOLIC REWIRING THROUGH PPAR/PGC1 MODULATION IMPROVES HEME/IRON-DRIVEN DEFECTIVE EFFEROCYTOSIS AND PROMOTES TISSUE DAMAGE RESOLUTION IN SICKLE CELL DISEASE
Authors: R. Sharma, S. Z. Vance, A. Antypiuk, D. Manwani, F. Vinchi
Journal: HemaSphere (2022)

Background: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is contributed by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme, which is released from oxidized hemoglobin upon hemolysis and acts as damage-associated molecular pattern to stimulate macrophage pro-inflammatory phenotypic switching through TLR4 signaling activation and ROS production. Heme-induced M1 pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to organ damage typically associated with this disease. Aims: While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties and whether this exacerbates tissue injury remain unexplored. Macrophage functions as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages, to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme strongly alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme and iron excess drives defective efferocytosis of apoptotic neutrophils in vitro, in cultured bone-marrow-derived macrophages, and in vivo, in mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the transcription factor PPARγ and its coactivator PGC1α. These changes lead to reduced expression of efferocytic receptors and impaired mitochondrial biogenesis as well as mitochondrial dysfunction, and is associated with metabolic skewing. This results in limited recognition and engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4/10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. We further demonstrate that impaired phagocytic capacity and tissue damage are improved by hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment in SCD mice through phenotypic and functional macrophage rewiring. Interestingly, defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients’ plasma and rescued by hemoglobin/heme scavenging via haptoglobin and hemopexin and PPARγ/PGC1α modulation via PPARγ agonist or IL-4. Summary/Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events.